Recent outbreaks of influenza highlight the need to rapidly produce and release adequate amounts of influenza vaccines to protect the general public from this disease, which has potentially deadly complications.
The standard assay for hemagglutinin (HA) content in inactivated influenza vaccines is based on single radial immunodiffusion (“SRID”) (refs. 1 & 2) which was recommended by the WHO in 1978 to replace tests based on agglutination of erythrocytes.
Although the SRID assay is well established, it is slow to perform, has poor dynamic range, is susceptible to considerable variability, and it can take a long time to prepare and calibrate the required specific anti-HA serum. As the influenza strains in vaccines change every season, this creates a bottleneck for influenza vaccine lot release because these reference reagents need to be prepared and calibrated anew for every strain change. This is particularly problematic in the case of an influenza pandemic where influenza vaccines need to be prepared as quickly as possible.
Another drawback of the SRID assay is that it may not reliably distinguish between immunogenically active forms of the influenza hemagglutinin (HA) antigen and those which are not as immunogenic because the antisera used in the assay may not be completely specific and may react with both forms, although it is generally thought that such antisera can be adjusted to preferentially recognize the native, immunogenic form in the SRID assay (ref. 125). As the immunogenicity (hence the immunoprotection) of an influenza vaccine is determined by the amount of immunogenically active HA, it is desirable for an assay to be able to specifically measure the immunogenically active form of HA.
Reference 3 suggests an alternative to a SRID assay in which ultrafiltration is followed by reverse phase high pressure liquid chromatography (RP-HPLC), and references 4 and 5 teach high pressure liquid chromatography (HPLC) based assays. References 6 and 7 developed quantitative mass spectrometry based assays. These assays could accurately quantify total HA and did not depend on strain-specific antisera, but failed to differentiate immunologically active HA from inactive HA. An ELISA assay was able to specifically quantify immunologically active HA but relied on generation of strain specific antibodies (ref. 8), which significantly increases the time needed before the vaccine can be released.